MitoBright LT Red
- High Intracellular Retention
- Long-Term Visualization of Mitochondria
- Provided as Ready-to-Use DMSO Solution
- Allows for Collagen-coated Plate
Product codeMT11 MitoBright LT Red
Unit size Price Item Code 20 ul ＄62.00 MT11-10 400 ul ＄123.00 MT11-12 400 ul x 3 ＄308.00 MT11-14
Dojindo Laboratories' Long-term mitochondrial staining reagent MitoBright LT series is widely used around the world. Here, we present some of the referenced publications, especially the ones in red font, indicating that MitoBright LT series can long-term stain mitochondria for up to 5 days.
Product Name Samples Observation time
Instrument Publications MitoBright LT Green Cell (U251) immediately Fluorescence microscope Pharmaceuticals MitoBright LT Green Yeast cell (Lipomyces starkeyi) immediately Fluorescence microscope Genes to Cells MitoBright LT Green Cell (HeLa) immediately Plate Reader Biomaterials MitoBright LT Green Cell (Naive CD4+ T cells) immediately Fluorescence microscope Cell Reports MitoBright LT Green Cell (CT26) immediately Flow Cytometer Journal of Radiation Research MitoBright LT Green Cell (C2C12) 5 Days Fluorescence microscope Polymers MitoBright LT Green Cell (BMM) 36 hrs or 3 Days Fluorescence microscope/
JCI Insights MitoBright LT Green Cells (mouse erythrocytes) immediately Fluorescence microscope Front. Cell. Infect. Microbiol. MitoBright LT Red Cell (SH-SY5Y) immediately Fluorescence microscope Free Radical Biol. Med. MitoBright LT Red Cell (MRC-5) immediately Fluorescence microscope The FEBS Journal MitoBright LT Red Cell (A11 cells/ P29 cells) 3 Days Fluorescence microscope BMC Mol. Cell Biol. MitoBright LT Deep Red Cell (HT-1080; MCF-10A; MCF-7; HCT-116 ) immediately Fluorescence microscope Small MitoBright LT Deep Red Cell (HT-1080; MCF-10A; MCF-7 ) 8 hrs Fluorescence microscope Advanced Therapeutics MitoBright LT Deep Red Cell (4T1) 24 hrs Fluorescence microscope Advanced Functional Materials MitoBright LT Deep Red Cell (SW982) immediately Fluorescence microscope GeneORDER
A mitochondrion is a multifunctional organelle not only involved in energy production in a cell, but other additional cellular functions. The active cycle of mitochondrial fusion and division induces morphological changes, which is called mitochondrial dynamics. Abnormalities in morphological control of mitochondria are associated with neurodegenerative diseases, metabolic disorders, aging, and so on. Therefore, the demand for long-term observation of mitochondrial dynamics has recently been increasing.
Methods for monitoring mitochondrial morphology, dynamics, and number are usually based on small fluorescent molecules or plasmid transfection techniques. The use of plasmids requires the target protein to be stably expressed, while small fluorescent molecules are widely used because they can simply be added to cells. Among commercially available small fluorescent molecules, those containing the chloromethyl moiety are commonly used. However, these dyes have some limitations, including short-term retention in cells, decreased fluorescence intensity in serum, and high background.
Dojindo’s MitoBright LT dyes overcome these limitations. MitoBright LT dyes are designed to exhibit mitochondria retention for long-term visualization. In addition, the MitoBright LT dyes show stronger fluorescence signals compared with other commercially available dyes that contain the chloromethyl moiety. The MitoBright LT dyes offer three different color options (Green, Red and Deep Red), and are provided as a ready-to-use DMSO solution. A working solution can easily be prepared in a single dilution step with growth medium or HBSS.
HeLa cells were washed with HBSS (Hank’s Balanced Salt Solution) and subsequently stained with each of MitoBright LTs or an existing reagent. The culture medium was replaced with serum-containing medium, and mitochondria were observed after 4 days of incubation. As a result, fluorescence intensity of an existing reagent decreased significantly after 4 days, but in MitoBright LT, fluorescence intensity remained unchanged and mitochondria were clearly observable. Moreover, after further incubation, we confirmed that MitoBright LT was retained in mitochondria even after 7 days.
Staining was performed with either MitoBright LT or an existing reagent in serum-containing or serum-free medium. In serum-containing medium, low fluorescence intensity was observed when using the existing reagent, however the cells stained with MitoBright LT experienced clear, resilient fluorescence of mitochondria
Jurkat cells (3.2×105 cells/mL) were suspended in RPMI medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin, plated on a 35cm dish, and incubated overnight at 37˚C in a 5% CO2 atmosphere. RPMI medium was removed and replaced with 5mL of MitoBright LT working solution (0.1μmol/L). The cells were then incubated for 30 minutes at 37˚C. The working solution was removed and the cells were washed twice with 5mL of RPMI medium. Fresh RPMI medium was poured into the dish and cells were passaged and analyzed every 2 days by flow cytometry.
Collagen-coated tissue culture plates are often used for examination of mitochondrial morphology because this imaging requires high magnifications.
The existing mitochondrial staining reagent bound to collagen, resulting with an increase in background staining, whereas the MitoBright LT series yielded clearly stained mitochondria.
Staining: HeLa cells were plated on a collagen coated tissue culture plate and cultured for 24 hours. The culture medium was removed, and the cells were washed with HBSS.
MitoBright LT Red working solution (100 nmol/L) was poured into the plate, the cells were incubated for 30 minutes. The solution was removed, and the cells were washed with HBSS. A fluorescence microscope was used for imaging of mitochondria.
Detection: Ex: 561 nm, Em: 560-620 nm
In mitochondria stained with an existing reagent, background staining was observed, but the mitochondria stained with MitoBright LT were clearly stained without having effect of background staining.
1. HeLa cells were seeded on a μ-slide 8 well plate and cultured at 37°C overnight in a 5% CO2 incubator.
2. The supernatant was removed and the cells were washed with serum-containing medium twice.
3. 0.1 μmol/l MitoBright LT (Green/Red/Deep Red) working solution (serum-containing medium) was added and the cells were incubated at 37°C for 15-60 min in the 5% CO2 incubator.
4. The supernatant was removed and the cells were washed with PBS twice.
5. 4% paraformaldehyde (PFA) solution was added and the cells were fixed at room temperature for 15 minutes.
6. The supernatant was removed and the cells were washed with PBS twice.
7. The cells were observed under a fluorescence microscope.
Figure 1. Fluorescent images of 4% PFA fixed HeLa cells (Stain then Fix)
Note: Cells should be fixed with paraformaldehyde (PFA). Fixation with methanol or other solvents extracts lipids and results in poor staining.
Note: The staining has low tolerance for permeabilization after fixation, and cannot be used with detergent.
(Triton X-100, NP-40 etc.)
|Microscope||A. M. Kimura, M. Tsuji, T. Yasumoto, Y. Mori, T. Oguchi, Y. Tsuji, M. Umino, A. Umino, T. Nishikawa, S. Nakamura, T. Inoue, Y. Kiuchi, M. Yamada, D. B. Teplow and K. Ono., " Myricetin prevents high molecular weight Aβ1-42 oligomer-induced neurotoxicity through antioxidant effects in cell membranes and mitochondria ", 2021, doi:10.1016/j.freeradbiomed.2021.05.019.|
|Microscope||Y. Takenaka, I. Inoue, T. Nakano, M. Ikeda and Y. Kakinuma, "Prolonged disturbance of proteostasis induces cellular senescence via temporal mitochondrial dysfunction and subsequent mitochondrial accumulation in human fibroblasts", 2021, doi:10.1111/febs.16249.|
(A11 cells/ P29 cells)
|Microscope||K. Takenaga, N. Koshikawa and H. Nagase, "Intercellular transfer of mitochondrial DNA carrying metastasis-enhancing pathogenic mutations from high- to low-metastatic tumor cells and stromal cells via extracellular vesicles", 2021, doi:10.1186/s12860-021-00391-5.|
Handling and storage condition
|Appearance：||Red purple liquid|
|-20°C, Protect from light|